The present invention relates to an adjuvanted vaccine, where lipid vesicles, particularly nonphospholipid lipid vesicles, serve as the adjuvant, together with methods of preparing the vaccine. Immunological adjuvants are the component of the vaccine which augment the immune response to the antigen. Immunological adjuvants function by, inter alia, attracting macrophages to the antigen and then to present that antigen to the regional lymph nodes and initiate an effective antigenic response. Adjuvants may also act as carriers themselves for the antigen. Many of the known immunological adjuvants, such as Freund""s complete adjuvant, alum, aluminum hydroxides, and Freund""s incomplete adjuvant, while effective at initiating the antigenic response, produce undesirable reactions in humans, such as inflammation at the point of injection. These side effects prevent use of such adjuvants in humans, and have led to the search for alternative immunological adjuvants.
Lipid vesicles are substantially spherical structures made of amphiphiles, e.g., surfactants or phospholipids. The lipids of these spherical vesicles are generally organized in the form of lipid bilayers, e.g., multiple onion-like shells of lipid bilayers which encompass an aqueous volume between the bilayers. Certain types of lipid vesicles have an unstructured central cavity which can be used to encapsulate and transport a variety of materials. Paucilamellar lipid vesicles, for example, have 2-10 peripheral bilayers surrounding a large, unstructured central cavity.
Until recently, liposome technology has been concerned mostly with vesicles composed of phospholipids. This is primarily because phospholipids are the principal structural components of natural membranes and, accordingly, lipid vesicles have been used as a model system for studying natural membranes. However, there are a number of problems associated with using phospholipids as synthetic membranes. Phospholipid liposomes placed in an in vivo environment are rapidly degraded. Moreover, phospholipids are labile and expensive to purify or synthesize. In addition, classic phospholipid liposomes are in the form of multilamellar as opposed to paucilamellar vesicles and have poor carrying capacities, especially for lipophilic materials, and have poor shelf lives unless lyophilized in the dark with antioxidants. Finally, phospholipids degrade too rapidly in vivo for most pharmaceutical or vaccine applications.
For these reasons, there is increasing interest in liposomes made of commercially available nonphospholipid amphiphiles (see, e.g.,U.S. Pat. No. 4,217,344, U.S. Pat. No. 4,917,951, and U.S. Pat. No. 4,911,928). These molecules have a hydrophilic head group attached to a hydrophobic xe2x80x9ctailxe2x80x9d and are derived from long chain fatty acids, long chain alcohols and their derivatives, long chain amines, and polyol sphingo- and glycerolipids. Commercially available amphiphile surfactants include, for example, the BRIJ(trademark) family of polyoxyethylene fatty ethers, the SPAN sorbitan fatty acid esters, and the TWEEN polyoxyethylene derivatives of sorbitan fatty acid esters, all available from ICI Americas, Inc. of Wilmington, Del. Paucilamellar vesicles containing such amphiphiles provide a high carrying capacity for water-soluble and water immiscible substances. The high capacity for water immiscible substances represents a unique advantage over classical phospholipid multilamellar liposomes.
Paucilamellar lipid vesicles may include a wide variety of phospholipids and nonphospholipid surfactants as their primary structural material. Paucilamellar lipid vesicles are substantially spherical structures made of materials having a high lipid content, preferably from nonphospholipid materials, which are organized in the form of lipid bilayers. The two to ten peripheral bilayers encapsulate an aqueous volume which is interspersed between the lipid bilayers and may also be encapsulated in the amorphous central cavity. Alternatively, the amorphous central cavity may be substantially filled with a water immiscible material, such as an oil or wax. Paucilamellar lipid vesicles have advantages as transport vehicles because large unstructured central cavity is easily adaptable for transport of large quantities of aqueous or oleaginous materials.
As described above, to stimulate a specific immune response, two components are required, namely the antigen or immunologicaly specific substance, and an adjuvant, the component augmenting the immune response to the antigen. Conventional adjuvants can serve as vehicles for the antigen, and as nonspecific immunological stimulants. The inventors have discovered that paucilamellar lipid vesicles are effective immunological adjuvants.
Accordingly, it is an object of the invention to provide an adjuvanted vaccine for immunizing against influenza, where paucilamellar lipid vesicles are the adjuvant.
Another object of the invention is to provide an adjuvanted vaccine to stimulate an immune response in a mammal, where the adjuvant is a nonphospholipid paucilamellar lipid vesicle which acts as a non-specific immune stimulator, an adjuvant/antigen carrier, or as a carrier of chemical adjuvants.
A further object of the invention is to provide a method of preparing adjuvanted vaccines useful in treating viral infections in mammals.
These and other objects and features of the invention will be apparent from the following description and from the claims.
The present invention features an adjuvanted vaccine, and methods for preparing an adjuvanted vaccine, preferably for immunizing against influenza, where the adjuvant is a lipid vesicle, and preferably is a nonphospholipid, paucilamellar lipid vesicle. The antigen may be encapsulated in the central cavity of the adjuvant, or mixed in solution with the adjuvant. Moreover, the adjuvant may carry a secondary adjuvant to further improve the immune response.
The antigen is preferably an influenza antigen and may comprise a formalin-inactivated whole virus, formalin-inactivated viral subunits, or an antigen produced by recombinant DNA techniques.
In one embodiment, the adjuvanted flu vaccine is prepared whereby the paucilamellar lipid vesicles, the preferred adjuvant, are prepared separately, and the adjuvant is then intermixed with the antigen. Alternatively, an adjuvanted vaccine can be prepared by forming paucilamellar lipid vesicles encapsulating the antigen.
The adjuvant in one embodiment of the invention is a paucilamellar lipid vesicle having about two to ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a large amorphous central cavity free of lipid bilayers. The lipid bilayers preferably have as their primary lipid component one or more of a the following nonphospholipid materials: polyoxyethylene fatty acid esters, polyoxyethylene fatty acid ethers, polyoxyethylene sorbitan esters, polyoxyethylene glyceryl mono- and diesters, glyceryl mono-and distearate, sucrose distearate, propylene glycol stearate, long chain acyl hexosamides, long chain acyl amino acid amides, long chain acyl amides, glyceryl mono-and diesters, dimethyl acyl amines, C12-C20 fatty alcohols, C12-C20 glycol monoesters, C12-C20 fatty acids, and mixtures thereof. More preferably, this mixture further contains at least one sterol selected from the group consisting of cholesterol, cholesterol derivatives, hydrocortisone, phytosterol, and mixtures thereof, a charge producing agent, and any lipid soluble or water soluble materials to be incorporated into the vesicles.
The vesicles of the present invention have a central cavity, carrying either water soluble materials or a water-immiscible oily solution, which can be used to encapsulate the antigen. The water-immiscible oily solution is made of materials which are both water immiscible and immiscible in the lipids used to form the bilayers. The water immiscible oily material found the amorphous central cavity may comprise soybean oil, squalene oil, squalane oil, sesame oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, sunflower oil, fish oils, petrolatum, avocado oil, triglyceride oils and fats, flavor oils, water insoluble vitamins, and mixtures thereof. These materials provide pharmacological benefits in addition to the benefits caused by the use of the particular lipids which form the bilayers.
The invention further features methods of producing adjuvanted vaccines. The adjuvant may comprise water or oil filled vesicles, e.g., vesicles having their amorphous central cavities filled with a water-immiscible oily solution, and these may be formed using either the xe2x80x9chot loadingxe2x80x9d technique disclosed in U.S. Pat. No. 4,911,928 or the xe2x80x9ccold loadingxe2x80x9d technique described in the U.S. Pat. No. 5,160,669, the disclosures of which are incorporated herein by reference. In either case, a lipid phase is formed by blending the nonphospholipid material, along with any sterols or lipophilic materials to be incorporated into the lipid bilayers, to form a homogenous lipid phase. In the xe2x80x9chot loadingxe2x80x9d technique, any water-immiscible oily material to be encapsulated in the vesicles is blended in the already formed lipid phase, forming a lipophilic phase. Oil-soluble or oil-suspendable antigens to be encapsulated within the vesicles are first dispersed in the oil. The term xe2x80x9cdispersedxe2x80x9d as used herein includes dissolution or forming a suspension or colloid to yield a flowable phase.
Once a lipophilic phase is made, it is blended with an aqueous phase (e.g., water, saline, or any other aqueous solution which will be used to hydrate the lipids), which may also contain an antigen, under shear mixing conditions to form the adjuvant. xe2x80x9cShear mixing conditionsxe2x80x9d, as used herein, means a shear equivalent to a relative flow of 5-50 m/s through a 1 mm orifice.
In the alternative, the vaccine can be incorporated into the amorphous central cavity of the adjuvant by the xe2x80x9ccold-loadingxe2x80x9d technique described in U.S. Pat. No. 5,160,669 to Wallach et al.
The scope and application of the invention will be apparent from the following detailed description.